Neural crest cell culture medium capable of participating in construction of regenerated teeth and culture method for inducing regenerated teeth

Abstract

The invention relates to a culture medium capable of participating in construction of neural crest cells of regenerated teeth and a culture method for inducing the regenerated teeth. More specifically, the culture medium is a DMEM / F12 culture medium containing or added with an N2 supplement, a B27 supplement, bFGF and EGF. The invention also relates to a method for culturing trunk neural crest cells by using the culture medium and a method for realizing full tooth regeneration by the trunk neural crest cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to a cell culture medium and its use. More specifically, the present invention relates to a cell culture medium and its use for culturing neural crest cells, a culture comprising the culture medium and neural crest cells, and a kit and its use for full tooth regeneration. The present invention also relates to a method for culturing trunk neural crest cells and a method for achieving full tooth regeneration by trunk neural crest cells. Background Art

[0002] The growth and development of teeth is a continuous and complex process. In this process, neural crest-derived ectomesenchymal cells interact with oral epithelial cells to form tooth germs, which further develop into complete teeth. Therefore, finding suitable seed cells to replace the epithelial cells and mesenchymal cells of tooth germs is one of the key issues in achieving tooth regeneration. Since scientists discovered dental pulp stem cells in 2000, dental pulp stem cells, periodontal ligament stem cells, and bone marrow stem cells have all been studied as replacements for mesenchymal cells, but these cells have failed to truly induce non-embryonic dental epithelium or non-dental epithelium to form tooth structures. On the other hand, after tooth eruption, adult dental epithelial cells are no longer available. Therefore, in recent years, researchers have developed a variety of seed cells that can replace tooth germ epithelial cells. These epithelial seed cells can form tooth-like structures together with embryonic dental mesenchymal cells. However, how to obtain odontogenic mesenchymal cells that can replace tooth germ mesenchymal cells (i.e., mesenchymal cells with odontogenic potential that can construct teeth together with dental or non-dental epithelium) remains a bottleneck problem to be overcome for more than ten years.

[0003] Since cranial neural crest cells can differentiate into dental mesenchymal cells in vivo, they can become important seed cells for in vitro tooth regeneration. In continuous research, researchers have successfully differentiated neural crest cells into odontoblasts or induced neural crest cells to differentiate into odontoblasts using bioengineering materials. The origin of odontogenic mesenchymal cells can be traced back to the embryonic period, when neural crest cells migrate ventrally and participate in the formation of various tissues and organs throughout the body, especially in the head region, where neural crest cells derive into a large number of mesenchymal cells and participate in the formation of tissues (Tan SS, Morrisskay G. Cell Tissue Res, 1985, 240 (2): 403-416; Santagati F, Rijli FM. Nat Rev Neurosci, 2003, 4 (10): 806-818; and Gross JB, Hanken J. Dev Biol, 2008, 317 (2): 389-400). Early studies have shown that the mesenchymal cells of tooth germs are mainly produced by the migration and differentiation of neural crest during development [Jheon AH, Schneider RA. J Dent Res, 2009, 88 (1): 12-21; and Miletich I, Sharpe PT. Neural crest contribution tomammalian tooth formation. Birth defects research Part C, Embryo today: reviews, 2004, 72 (2): 200-212]. Therefore, it is expected that the regeneration of tooth germ mesenchyme can be achieved by obtaining neural crest cells in vitro. During craniofacial development, neural crest cells form different compartments during the migration and formation process due to different proliferation, namely the branchial arches. Trallnor et al. showed that in the early stages of craniofacial development, cranial neural crest cells derived from the neuroectoderm cells of the posterior midbrain and forebrain and anterior posterior brain occupied most of the first branchial arch mesenchyme and were located in the maxillary and maxillary processes [Trainor P, Krumlauf R. Nature cell biology, 2000, 2 (2): 96-102]. Subsequently, related studies further confirmed the role of neural crest cells in mammalian tooth development. The researchers recombined mouse neural crest and odontogenic epithelium and transplanted them into the mouse anterior eye socket for culture, which resulted in the formation of teeth, providing direct evidence for the role of neural crest in mammalian tooth formation.This research result also shows that tooth development mainly originates from a series of epithelial-mesenchymal interactions between mandibular epithelium and neural crest-derived ectomesenchyme [Thesleff I, Sharpe P. Mechanisms of development, 1997, 67 (2): 111-123., and Jernvall J, Thesleff I. Mechanisms of development, 2000, 92 (1): 19-29. The study of Imai et al. also showed that ectomesenchymal cells derived from neural crest produce odontogenic mesenchymal cells, which then differentiate into odontoblasts that can secrete dentin [Imai H, Osumi-Yamashita N, Ninomiya Y, et al. Dev Biol, 1996, 176 (2): 151-165.]. Moreover, mutations in neural crest transcription factors can lead to abnormal development of the skull and teeth [Satokata I, Maas R. Nature genetics, 1994, 6 (4): 348-356; and Peters H, Neubuser A, Kratochwil K, et al. Genes & development, 1998, 12 (17): 2735-2747]. Therefore, the above research results all indicate that neural crest cells are of great significance to the formation of tooth germ mesenchyme and the development of tooth organs. At the same time, this research conclusion also has very important guiding value for how to achieve tooth regeneration from the perspective of tooth germ mesenchyme.

[0004] Application of neural crest cells in tooth regeneration: Mitsiadis et al. transplanted mouse embryo neural crest tissue into the neural crest of chick embryos at the corresponding period and found that the first branchial arch of the chick embryo had regenerated tooth germ structure after a few days [Mitsiadis TA, Cherad Y, Sharpe P, et al. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100 (11): 6541-6545.]. Chung et al. transplanted HA / TCP as a scaffold, and transplanted the migrated cranial neural crest cells of embryonic day 10.5 and the cells of tooth germ of embryonic day 13.5 into the subcutaneous tissue of nude mice and found that neural crest cells can generate bone marrow matrix and support the development of tooth germ. Therefore, the migrated cranial neural crest cells are not only of great significance for the formation of cranial tissues, but also play an important role in the development of tooth organs [Chung IH, Yamaza T, Zhao H, et al. Stem cells, 2009, 27 (4): 866-877.]. Domestic scholars' research also shows that after induction in vitro, cranial neural crest cells can reflect the characteristics of odontoblasts to a certain extent, and after being implanted into the subcutaneous tissue of nude mice with collagen-chitosan three-dimensional gel scaffolds, dentin-like structures will be generated [Jiang HB, Tian WD, Liu LK, et al. Cell biology international, 2008, 32 (6): 671-678.]. These all indicate that cranial neural crest cells are an ideal seed cell source for tooth differentiation and regeneration.

[0005] The present invention successfully separated and cultured the neural crest cells of the trunk, which could form a complete tooth-like structure with the epithelial components after multiple passages, expanding the range of neural crest cells that can be used for full tooth regeneration, and proposed a new type of mesenchymal candidate cells that can be used for full tooth regeneration. Summary of the invention

[0006] Currently, all the successfully constructed regenerated teeth known contain epithelial or mesenchymal parts derived from tooth germs, which greatly restricts the output of regenerated teeth and their clinical transformation and application. How to obtain odontogenic mesenchymal cells that can replace tooth germ mesenchymal cells (i.e., mesenchymal cells with odontogenic potential that can construct teeth together with odontogenic or non-odontogenic epithelium) is still one of the key issues in achieving tooth regeneration. Tooth germ mesenchymal cells eventually develop into dentin, dental pulp and periodontal tissues, which contain mature odontogenic cells such as dental pulp (stem) cells and periodontal ligament (stem) cells. However, these cells have completely lost the cell population that induces non-induced odontogenic fate.

[0007] In this regard, the present invention successfully separated and cultivated neural crest cells of the trunk, which can form a complete tooth-like structure with epithelial components after multiple passaging, expanding the range of neural crest cells that can be used for full tooth regeneration, and proposing a new type of mesenchymal candidate cells that can be used for full tooth regeneration.

[0008] In one aspect, the present invention relates to a cell culture medium, which is a DMEM / F12 medium containing or supplemented with N2 supplement, B27 supplement, bFGF and EGF. In the culture medium, bFGF is present in an amount of 1 ng / μL-100 ng / μL, such as 5 ng / μL, 10 ng / μL, 20 ng / μL, 30 ng / μL, 40 ng / μL, 50 ng / μL, 60 ng / μL, 80 ng / μL, preferably 20 ng / μL. In the culture medium, EGF is present in an amount of 1 ng / μL-100 ng / μL, such as 5 ng / μL, 10 ng / μL, 20 ng / μL, 30 ng / μL, 40 ng / μL, 50 ng / μL, 60 ng / μL, 80 ng / μL, preferably 20 ng / μL.

[0009] N2 supplement and B27 supplement are generally provided in the form of concentrated solution and added to the cell culture medium of the present invention, for example, the concentration degree can be 50×, 100× or 200×. Taking 200× concentration degree as an example, in the culture medium, the content of 200×N2 supplement can be 0.1% volume-1% volume, for example, 0.2% volume, 0.3% volume, 0.4% volume, 0.6% volume, 0.8% volume, preferably 0.5% volume. In addition, taking 100× concentration degree as an example, in the culture medium, the content of 100×B27 supplement can be 0.2% volume-2% volume, for example, 0.3% volume, 0.4% volume, 0.5% volume, 0.6% volume, 0.8% volume, 1.2% volume, 1.4% volume, 1.5% volume, 1.6% volume, 1.8% volume, preferably 1% volume. It is understood by those skilled in the art that the above-mentioned supplements are added to the culture medium in an appropriate amount or content according to the concentration degree.

[0010] In one aspect, the cell culture medium can be used to culture neural crest cells, such as trunk neural crest cells. The present invention relates to the use of the cell culture medium for culturing trunk neural crest cells. In this aspect, the neural crest cells are cultured to proliferate in vitro and maintain the ability to participate in tooth induction.

[0011] In one aspect, the present invention relates to a cell culture comprising the above-mentioned cell culture medium of the present invention and trunk neural crest cells.

[0012] In one aspect, the present invention relates to a kit comprising a first cell culture medium and a second culture medium, wherein the first cell culture medium is a DMEM / F12 culture medium containing or supplemented with N2 supplement, B27 supplement, bFGF and EGF, and the second culture medium is a high-glucose DMEM culture medium containing or supplemented with FBS. In the second culture medium, the content of FBS in the high-glucose DMEM culture medium can be 1%-20%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 11%, 12%, 14%, 15%, 16%, 18%, preferably 10%. The kit can be used for full tooth regeneration, specifically for achieving full tooth regeneration by neural crest cells such as trunk neural crest cells. The first cell culture medium and the second culture medium can be placed separately, for example, in separate containers.

[0013] In one aspect, the present invention relates to the use of the above kit for full tooth regeneration, wherein the first cell culture medium is used to culture trunk neural crest cells, and the second culture medium is used to culture a mixture of trunk neural crest cells cultured in the first culture medium and tooth germ epithelial cells or tissues.

[0014] In one aspect, the present invention relates to a method for culturing trunk neural crest cells, comprising the step of culturing trunk neural crest cells in the above-mentioned cell culture medium. The present invention may also relate to a method for culturing trunk neural crest cells to expand in vitro and maintain the ability to participate in odontogenesis, comprising the step of culturing trunk neural crest cells in the above-mentioned cell culture medium to expand in vitro and maintain the ability to participate in odontogenesis. In this aspect, the cell culture medium is the above-mentioned DMEM / F12 culture medium containing or supplemented with N2 supplement, B27 supplement, bFGF and EGF. In the step of culturing trunk neural crest cells, the trunk neural crest cells are cultured at 37°C and 5% CO2, preferably incubated for 48 hours; the culture medium is replaced every two days; and / or the cells are passaged once every 3 to 4 days at a ratio of 1:3. The method may also include the step of centrifuging and collecting the trunk neural crest cells cultured at P3-P5 (P3, P4 and / or P5) generations and incubating them to aggregate the cells. In this step, 1.0 x 10 5 Cells, centrifuged at 4 degrees, at 6600 rpm for 4 minutes. In this step, incubated at 37°C for 1-3 hours to allow the cells to aggregate. Specifically, in this step, 400 microliters are retained in each tube after centrifugation, placed in a 37 degree incubator, and incubated for 1-3 hours to promote cell aggregation. In this aspect, trunk neural crest cells are cultured and expanded in vitro and maintain the ability to participate in tooth induction.

[0015] In one aspect, the present invention relates to a method for achieving full tooth regeneration by trunk neural crest cells. The method comprises a) a step of culturing trunk neural crest cells as described above; b) a step of aggregating the cultured trunk neural crest cells; and c) a step of recombining the cell agglomerates obtained from step b) with tooth germ epithelial cells or tissues to obtain a recombinant. The method may also include d) a step of transplanting the recombinant obtained from step c) into a host. In step a), trunk neural crest cells are cultured to expand in vitro and maintain the ability to participate in tooth induction. In step a), trunk neural crest cells are cultured using DMEM / F12 medium containing or supplemented with N2 supplement, B27 supplement, bFGF and EGF. In step b), the cells obtained from step a) are agglomerated. In step b), trunk neural crest cells cultured at generations P3-P5 (P3, P4 and / or P5) are centrifuged and collected and incubated to agglomerate the cells. For example, incubate at 37°C for 1-3 hours to allow the cells to aggregate. In step c), the recombination is achieved by culturing the cell aggregates with tooth germ epithelial cells or tissues in a high-glucose DMEM medium containing or supplemented with FBS, preferably overnight in gas-liquid two-phase culture. The content of FBS in the high-glucose DMEM medium can be 1%-20%, for example 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 11%, 12%, 14%, 15%, 16%, 18%, preferably 10%. In step c), the molar germs are isolated from the mandibular bones of mammals, incubated with Dispase II, digested and washed with serum-containing medium, and then the tooth germ epithelium and tooth germ mesenchyme are separated to obtain tooth germ epithelial cells or tissues. More specifically, in step c), the molar germs are isolated from the mandibular bones of mammals and incubated at 37°C for 35 minutes with 0.75 mg / mL DispaseII. In step d), the obtained recombinant can be transplanted under the renal capsule of the host. In this aspect, the trunk neural crest cells are expanded in vitro through culture and maintain the ability to participate in the induction of odontogenesis.

[0016] In one aspect, the tooth germ epithelial cells or tissues are tooth germ epithelial cells or tissues that do not have the ability to induce tooth formation after the tooth germ bud stage.

[0017] In the method of the present invention, the culturing method is performed in vitro.

[0018] In the present invention, trunk neural crest cells can be obtained by the following steps: the neural tube and surrounding tissues with a thin layer of tissue around them are cultured by patch method, and the neural tube and its surrounding tissues are taken from the trunk position of the mammalian embryo; the remaining tissue blocks are removed the next day and cultured continuously; and the trunk neural crest cells crawled out by patch method are collected. In one aspect, the cells cultured by patch method are digested with dispase, such as 1.5 mg / mL dispase, at 37°C for 10 minutes, and washed. The washed cells can be inoculated on a 6-well plate coated with matrix gel.

[0019] In one aspect, trunk neural crest cells are expanded in vitro and maintain the ability to participate in tooth induction by culturing in the medium of the present invention.

[0020] In the present invention, the cell or tissue may be derived from a mammal. The host may be a mammal. The cell, tissue and host may be derived from the same mammal. The mammal may be a cow, sheep, pig, dog, cat, mouse, rat, guinea pig or monkey.

[0021] As used herein, "N2 supplement" or "N2 additive" is a chemically defined serum-free supplement based on Bottenstein's N-1 formulation. N2 supplement can be used to support the growth and expression of primary neuroblastoma and postmitotic neurons derived from the peripheral and central nervous systems. N2 supplement is commercially available from Gibco, 17502-048.

[0022] In this article, "B27 supplement" or "B27 additive" is the most commonly cited neuronal cell culture supplement, which is an optimized serum-free supplement used to support low-density or high-density growth and short-term or long-term viability of embryonic, postnatal, and adult hippocampal and other central nervous system neurons. B27 supplement is commercially available as Gibco, 17504-044.

[0023] "bFGF" refers to basic fibroblast growth factor. "EGF" refers to epidermal growth factor. "FBS" refers to fetal bovine serum.

[0024] The full name of “DMEM / F12 medium” is “Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 medium”, which is a medium prepared by mixing DMEM medium and F12 medium in a 1:1 ratio. In one example, the mixed medium may contain L-glutamine and hydroxyethylpiperazineethanesulfonic acid (HEPES). For example, the DMEM / F12 medium may be commercially available as Hyclone, SH30023.01.

[0025] "DMEM medium" also known as "Dulbecco's Modified Eagle Medium" or "Dulbecco's Modified Eagle Medium" is a widely used basal medium used to support the growth of many different mammalian cells. DMEM medium contains various amino acids and glucose and is developed based on MEM medium.

[0026] “High glucose DMEM medium” refers to a DMEM medium with a glucose content of about 4500 mg / L.

[0027] In the present invention, when referring to cell culture and passage, P represents the passage number. For example, P3-P5 refers to the 3rd to 5th passage cells.

[0028] Compared with invertebrates, vertebrates have more advanced physiological structures and more complex physiological functions. The fundamental reason for this difference is that vertebrates have a group of cells that can migrate and differentiate, which are called neural crest. Neural crest cells can differentiate into a variety of cells, including neurogenic cells, osteoblasts, melanocytes, and play an important role in the formation of craniofacial structures. Neural crest cells can differentiate into a variety of cells, including neurogenic cells, osteoblasts, melanocytes, and play an important role in the formation of craniofacial structures. Because neural crest cells have multiple differentiation potentials, they are also called the fourth germ layer. On embryonic day 6 (E6), the developing notochord and adjacent mesenchyme induce the ectoderm on its surface to form a neural plate, which gradually closes to form a neural tube. Neural crest cells originate between the neural tube and the surface ectoderm, forming a cell band along the head and tail of the embryo, and then gradually undergo epithelial-mesenchymal transformation. Due to different locations, neural crest cells can be divided into five types: skull, heart, vagus nerve, trunk, and sacrum. They can migrate and differentiate under the regulation of signal pathways, and eventually form different tissues and organs.

[0029] During craniofacial formation, a small group of cranial neural crest cells can differentiate into dental mesenchymal cells under the influence of the surrounding environment and extracellular signals. The present invention unexpectedly found that the neural crest cells of the trunk can also differentiate into dental mesenchymal cells, and can be combined with epithelial components to form a complete tooth-like structure including enamel, dentin and pulp. The present invention is the first to confirm that the neural crest cells of the trunk, like the neural crest cells of the head and neck, have the potential to develop into teeth.

[0030] The present invention successfully separated and cultured the neural crest cells of the trunk, which can form a complete tooth-like structure with the epithelial components after multiple passaging. Studies have found that the neural crest cells can be used as candidate cells of mesenchymal properties for full tooth regeneration, providing a new source of seed cells and a new research scheme for full tooth regeneration research, which is conducive to promoting the construction research of biological regenerative teeth. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The process of obtaining and culturing neural crest cells and recombining them with tooth germ epithelial cells or tissue to achieve full tooth regeneration is shown.

[0032] Figure 2 The process of obtaining and culturing trunk neural crest cells from embryonic mice and the results of each stage in Example 1 are shown. Figure 2 A and 2B show intact embryonic mice; Figure 2 C shows the neural tube and its surrounding tissues at the trunk position removed from the embryonic mouse; Figure 2 D shows P0 cells cultured in the medium of the present invention for 3 days; Figure 2 E shows P3 cells cultured in the medium of the present invention for 3 days; Figure 2 F shows P5 cells cultured in the medium of the present invention for 3 days.

[0033] Figure 3 The observation results of the regenerated complete tooth obtained in Example 1 are shown. Figure 3 A shows: a bright field image of the product of the transplant in Example 1 after three weeks under the renal capsule (tooth-like structure); Figure 3 B shows: the product separated from under the renal capsule (tooth-like structure); Figure 3 C shows: the tooth structure diagram of the product of the transplant under the renal capsule in Example 1 after three weeks of decalcification and sectioning, and HE staining. DETAILED DESCRIPTION

[0034] Example 1

[0035] The process of this embodiment is as follows Figure 1 As shown:

[0036] (1) From embryonic mice at 9.5 gestation (e.g. Figure 2 A and Figure 2 B) Isolation and culture of trunk neural crest cells

[0037] - Pregnant ICR mice were killed and the neural tube and surrounding tissues (such as Figure 2 C Trunk cutting position);

[0038] - The neural tube and surrounding tissues with a thin layer of tissue around them were cultured by patch method (such as Figure 2 D)

[0039] -Remove the remaining tissue pieces the next day and continue culturing;

[0040] - Collect cells crawling out of the patch method: digest with dispase (1.5 mg / mL, Gibco, 1626457) at 37°C for 10 minutes, wash and inoculate on a 6-well plate coated with matrix gel (Corning, 100×, 354277);

[0041] - Culture medium: DMEM / F12 (Hyclone, SH30023.01)

[0042] +200×N2 supplement (Gibco, 17502-048)

[0043] +100× B27 supplement (Gibco, 17504-044)

[0044] +20ng / μL bFGF

[0045] +20ng / μL EGF

[0046] After incubation at 37 °C and 5% CO2 for 48 h, the culture medium was changed every two days.

[0047] - Subculture at a 1:3 ratio every 3-4 days.

[0048] (2) Collecting neural crest cells in culture to form cell aggregates

[0049] - Collect P3-P5 cells (such as Figure 2 E and Figure 2 F), counting and centrifugation, 1.0 x 105 cells per 1.5 ml centrifuge tube, centrifugation at 4 degrees, 6600 rpm, 4 minutes;

[0050] - After centrifugation, keep 400 μl in each tube and incubate in a 37°C incubator for 1-3 hours to allow cells to aggregate.

[0051] (3) Recombination and transplantation

[0052] - Molar germs were isolated from the mandibles of E14.5 mice and incubated with 0.75 mg / mL Dispase II at 37°C for 35 minutes;

[0053] -After terminating digestion and washing with serum-containing medium, separate the tooth germ epithelium and tooth germ mesenchyme with a needle, and take the tooth germ epithelium for later use;

[0054] - The cell mass after incubation was taken out and recombined with the isolated E14.5 tooth germ epithelium, and cultured overnight in an air-liquid two-phase (Trowell-type system) (culture medium: high-glucose DMEM + 10% FBS);

[0055] - transplanting the recombinant sample under the host's renal capsule;

[0056] -3 weeks later, the host mice were dissected to examine the calcified tissue formed by the recombinant samples.

[0057] The results are as follows Figure 3 A. Figure 3 B and Figure 3 As shown in Figure C, this study successfully isolated and cultured neural crest cells from the trunk, which can form a complete tooth-like structure with the epithelial component after multiple passages. The study found that it can be used as a candidate cell of mesenchymal nature for full tooth regeneration, providing a new source of seed cells and a new research program for full tooth regeneration research, which is conducive to promoting the construction of biological regenerative teeth.

[0058] Example 2

[0059] Statistics of tooth formation rate

[0060] In each biological experiment (Batch), cells from any generation of P3, P4 and P5 were selected to carry out the tooth germ epithelium recombination and transplantation experiments described in Example 1, and the tooth formation rate was detected after 3 weeks.

[0061] Excluding the initial preliminary experiment, the dorsal neural crest-related experiments were conducted for 5 rounds, and the tooth formation rates are shown in Table 1 . The tooth formation rates shown in the experimental results, such as 2 / 15, represent that 2 out of 15 samples formed tooth-like structures.

[0062] Table 1. The tooth formation rate of full tooth regeneration from trunk neural crest cells in five rounds of biological experiments.

[0063] Trunk neural crest cells percentage Batch 1-P3 2 / 15 Batch 2-P3 4 / 23 Batch 3-P4 6 / 12 Batch 4-P4 2 / 12 Batch 5-P5 3 / 15 total 17 / 77(22.08％)

[0064] Using the same approach, full tooth regeneration from cranial neural crest (head and neck neural crest cells) achieved similar tooth formation rates.

[0065] The above embodiments are only used to illustrate the present invention, but not to limit the present invention. Ordinary technicians in the relevant technical field can make various changes and modifications without departing from the scope of the present invention. Therefore, all equivalent technical solutions should also fall within the scope of the present invention.

Claims

1. A cell culture medium, which is a DMEM / F12 medium containing or supplemented with N2 supplement, B27 supplement, bFGF and EGF.

2. The cell culture medium according to claim 1, wherein the culture medium comprises or is supplemented with 1 ng / μL-100 ng / μL, preferably 20 ng / μL, of bFGF, and / or 1 ng / μL-100 ng / μL, preferably 20 ng / μL of EGF.

3. The cell culture medium of claim 1 or 2, wherein the culture medium comprises or is supplemented with 0.1% vol.-1% vol., preferably 0.5% vol., of 200×N2 supplement and / or 0.2% vol.-2% vol., preferably 1% vol., of 100×B27 supplement.

4. Use of the cell culture medium according to any one of claims 1 to 3 for culturing trunk neural crest cells.

5. A cell culture comprising the culture medium of any one of claims 1 to 3 and trunk neural crest cells.

6. A kit comprising the culture medium according to any one of claims 1 to 3 as a first culture medium, and a high-glucose DMEM culture medium containing or supplemented with FBS as a second culture medium, preferably the high-glucose DMEM culture medium contains or supplemented with 10% FBS.

7. Use of the kit of claim 6 for whole tooth regeneration, wherein the first culture medium is used to culture trunk neural crest cells, and the second culture medium is used to culture a mixture of trunk neural crest cells cultured in the first culture medium and tooth germ epithelial cells or tissues.

8. A method for culturing trunk neural crest cells, comprising the step of culturing the trunk neural crest cells in the culture medium according to any one of claims 1 to 3.

9. The method of claim 8, wherein in the step of culturing trunk neural crest cells, The trunk neural crest cells are cultured at 37°C and 5% CO2, preferably for 48 hours; Change the culture medium every two days; and / or Subculture every 3 to 4 days at a 1:3 ratio.

10. The method of claim 8 or 9, further comprising the step of collecting the cultured trunk neural crest cells, such as the cultured trunk neural crest cells at any passage from P3 to P5, by centrifugation and incubating the cells to aggregate.

11. The method of claim 10, wherein the incubation is performed at 37°C for 1-3 hours to allow the cells to aggregate.

12. A method for achieving full tooth regeneration using trunk neural crest cells, comprising: a) a step of culturing trunk neural crest cells as defined in claim 8 or 9; b) a step of agglomerating the cultured trunk neural crest cells as defined in claim 10 or 11; c) a step of recombining the cell aggregate obtained in step b) with tooth germ epithelial cells or tissues to obtain a recombinant; and d) Optionally, the step of transplanting the recombinant obtained in step c) into a host.

13. The method of claim 12, wherein in step c), the recombination is achieved by culturing the cell aggregates with tooth germ epithelial cells or tissues in a high-glucose DMEM medium containing or supplemented with FBS, preferably culturing overnight in both air-liquid phases.

14. The cell culture medium of any one of claims 1 to 3, the use of claim 4 or 7, the cell culture of claim 5, the kit of claim 6, or the method of any one of claims 8 to 13, wherein the cells, tissues or hosts are derived from the same mammal, such as selected from cattle, sheep, pigs, dogs, cats, mice, rats, guinea pigs or monkeys.

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